Hepatitis C virus (which may be simply referred to as “HCV” hereinafter) was discovered and identified as a causative virus of non-A and non-B hepatitis by Choo et al. in 1989 (Choo, Q L. et al., Science, 244: 359-362, 1989). HCV infection is a major cause for the progression with persistent infection from chronic hepatitis to cirrhosis and then hepatic cancer. It is said that there are approximately 170,000,000 HCV patients in the world and approximately 2,000,000 HCV patients in Japan. The major route of infection is transmission through blood. Since the screening of blood for transfusion was made available, the number of people newly infected with HCV dramatically decreased in Japan. However, it is considered that many virus carriers still remain.
At present, HCV is mainly treated with the use of PEG-interferon or with the use of PEG-interferon in combination with an antiviral drug (i.e., ribavirin). HCV is currently classified into 6 different genotypes, and HCV of genotype 1b and of genotype 2a are major types in Japan. In the case of HCV of genotype 1b, in particular, viruses cannot be completely eliminated from the body via administration of interferon and ribavirin, and therapeutic effects are insufficient. Accordingly, development of novel antiviral drugs or vaccines aimed at preventing virus carriers from causing the disease to develop or at eliminating viruses has been awaited.
The lack of effective animals that reflect virus infection besides chimpanzees and the lack of an effective in vitro virus culture system had been impediments to the development of therapeutic agents of HCV. In recent years, HCV replicon systems that enable evaluation of HCV-RNA replication have been developed (Lohmann, V. et al., Science., 285: 110-113, 1999), and such systems resulted in important progress as a system for screening for HCV inhibitors associated with the inhibition of virus replication.
HCV is a single-stranded (+) RNA virus having a genome length of approximately 9.6 kb, which has a gene encoding a precursor protein converted into 10 types of virus proteins (i.e., Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins) upon post-translational cleavage by proteases. The replicon system was prepared by recombining the translation region of the HCV structural protein with a drug-resistant gene and inserting IRES of the encephalomyocarditis virus (EMCV) downstream thereof. RNA replication is observed in cells into which such recombinant RNA has been introduced. Even when a full-length genome RNA having the HCV structural protein region has been introduced into cells, however, release of virus particles into the culture solution has not been observed (Pietschmann, T. et al., J. Virol., 76: 4008-4021, 2002).
Recently, the HCV JFH-1 strain of genotype 2a isolated from a patient with fulminant hepatitis was discovered by Wakita et al., and the strain was found to be released as infectious virus particles in a culture medium of Huh-7 cells (hepatic cancer cell lines) (WO 05080575A1 and Wakita, T. et al., Nat. Med., 11: 791-796, 2005). This in vitro culture system for infectious HCV particles is expected to serve as a useful screening tool in the development of anti-HCV agents and to be an effective means for preparation of HCV vaccines. Research on HCV particle production in in vitro culture systems has made progress, and the HCV genome capable of virus particle production was found to be chimeric HCV of the JFH-1 strain and an HCV strain other than the JFH-1 strain. Such chimeric HCV can be prepared by recombining the structural genes of the JFH-1 genome (i.e., Core, E1, E2 and p7 protein coding sections) with structural genes of other HCV strains.
As chimeric HCV of the JFH-1 strain and an HCV strain other than the JFH-1 strain, the chimeric HCV of the J6CF strain (genotype 2a) and the JFH-1 strain (Lindenbach, B. D. et al., Science, 309: 623-626, 2005), the chimeric HCV of the H77 strain (genotype 1a) and the JFH-1 strain (WO 06096459A2 and MinKyung, Y. et al., J. Virol., 81: 629-638, 2007), and the chimeric HCV of the S52 strain (genotype 3a) and the JFH-1 strain (Gottwein, J M et al., Gastroenterology 133: 1614-1626, 2007) are known.
Pietschmann, T. et al. (Proc. Natl. Acad. Sci. U.S.A., 103: 7408-7413, 2006) discloses that the amount of viruses produced from chimeric HCV of the J6CF structural gene and the JFH-1 non-structural gene is the highest and the amount of infectious virus particles produced from chimeric HCV of the Con1 strain of genotype 1b and the JFH-1 strain is one tenth the former amount. As other chimeric HCV of a genotype 1b strain and the JFH-1 strain, WO 06022422A1 discloses that a genome (full-length genome replicon RNA) in which a region encoding the structural proteins of the TH strain is recombined with that of the JFH-1 genome, and a drug-resistant gene is inserted into a site upstream of such coding region was produced, the resulting genome is introduced into the Huh-7 cell, and then a drug-resistant strain is obtained, and infectious virus particles are produced into the culture supernatant, although productivity thereof is not clearly described.
Under such circumstances, concerning genotype 1b for which the possibility of attaining a complete remission with current therapy techniques is small and of which the number of patients is large, there is a need for development of a method for producing HCV particles that can produce large quantities of infectious virus particles with the structure of genotype 1b, and that can be cultured in a persistent infection system.